Crystalline salts of (4s,4as,5ar,12as)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylic acid amide and methods of using the same

ABSTRACT

A crystalline mono hydrochloride salt of (4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylic acid amide is disclosed having improved stability. In addition, a crystalline mono mesylate salt and crystalline mono sulfate salt of (4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylic acid amide are also disclosed having improved stability. A pharmaceutical composition containing the crystalline salts and methods of treating inflammatory skin disorders and bacterial infections comprising administering the crystalline salts are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 61/485,179, filed May 12, 2011, the content of which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The instant disclosure relates to crystalline mono hydrochloride, monomesylate, and mono sulfate salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, and methods of using the same. More specifically, thedisclosure relates to crystalline mono hydrochloride, mono mesylate, andmono sulfate salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide having improved stability over tetracycline compounds knownin the art. In addition, the instant disclosure relates topharmaceutical compositions comprising the crystalline monohydrochloride, mono mesylate, or mono sulfate salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, and methods of treating acne, rosacea or gram positivebacterial infections using the crystalline mono hydrochloride, monomesylate, or mono sulfate salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

BACKGROUND OF THE INVENTION

Tetracyclines are known “broad spectrum” antibiotics and have becomewidely used for therapeutic purposes. Tetracyclines have been found tobe highly effective pharmacologically against rickettsiae; a number ofgram-positive and gram-negative bacteria; and the agents responsible forlymphogranuloma venereum, inclusion conjunctivitis, and psittacosis. Thefirst use of tetracycline antibiotics dates as far back as 1948.Examples of pharmaceutically active tetracycline and tetracyclineanalogue compositions may be found in U.S. Pat. Nos. 2,980,584;2,990,331; 3,062,717; 3,165,531; 3,454,697; 3,557,280; 3,674,859;3,957,980; 4,018,889; 4,024,272; and 4,126,680. Tetracyclines may alsobe used to treat inflammatory skin disorders, including dermatitis,psoriasis, pyoderma gangrenosum, acne and rosacea.

Acne vulgaris, also referred to as acne, is both an inflammatory skindisorder and a bacterial infection. It is a disorder resulting fromhormones affecting the sebaceous glands, which leads to plugged poresand outbreaks of lesions, or pimples. Acne is the most common skindisease in the United States, affecting nearly 17 million people. Severeacne can lead to disfiguration, and permanent scarring.

Acne is described as a disorder of the pilosebaceous units (PSUs). Foundover most of the body, PSUs consist of sebaceous glands, which make anoily substance that normally empties onto the skin surface through theopening of the follicle, also called a pore. When the pore is plugged,the mixture of oil and cells allows bacteria that normally live on theskin to grow in the plugged follicles, which produce chemicals andenzymes and attract white blood cells that cause inflammation. Theplugged follicle breaks down, the sebum, shed skin cells and bacteriadisseminate into the nearby tissues, leading to lesions or pimples.

Acne is commonly treated with systemic antibiotics, includingtetracyclines, to reduce the growth of bacteria. Efficacy is thought tobe due to an effect on Propionibacterium acnes (P.acnes) as well as theintrinsic anti-inflammatory properties of these antibiotics.Propionibacterium acnes is a relatively slow growing, typicallyaerotolerant anaerobic gram positive bacterium (rod) that is linked toacne. Tetracyclines are known to be effective in killing P.acnes andother bacteria and have been used to treat acne because of theirantibacterial and anti-inflammatory properties.

Rosacea is a skin disorder characterized by facial redness, mainlyaffecting individuals of north western European descent. Early symptomsof rosacea include redness on the chin, nose, skin or forehead; smallvisible blood vessels on the face; bumps or pimples on the face; andwatery and irritated eyes. Although the causes of rosacea are poorlyunderstood, systemic antibiotics, such as tetracyclines, are commonlyprescribed for the treatment of rosacea, due to both theiranti-inflammatory and antibacterial properties.

After the widespread use of tetracyclines for both major and minorillnesses and diseases led to resistance to these antibiotics,substituted tetracycline compounds were developed to treat bacterialinfections, inflammation, neoplasms, and other conditions. The term“tetracycline compound” includes many compounds with a similar ringstructure to tetracycline. Examples of these tetracycline compoundsinclude: chlortetracycline, doxycycline, minocycline, oxytetracycline,demeclocycline, methacycline, sancycline, chelocardin, rolitetracycline,lymecycline, apicycline; clomocycline, guamecycline, meglucycline,mepylcycline, penimepicycline, pipacycline, etamocycline, penimocycline.For example, substituted tetracycline compounds have been disclosed inWO 2008/079339 and WO 2008/079363.

One substituted tetracycline compound is(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, described in U.S. Patent Application Publication Nos.2008/0312193 and 2010/0305072. The free base of this compound has provenunstable for use as an active pharmaceutical ingredient. In addition,while those skilled in the art have attempted to synthesize a salt ofthis compound previously, only amorphous salts have been produced andthese amorphous salts have shown only minimal improved stability overthe free base. Accordingly, there exists a need in the art for improvedstability of this substituted tetracycline compound.

The present invention is directed to the novel crystalline monohydrochloride, mono mesylate, and mono sulfate salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, which exhibit superior stability over the free base andpreviously known salts thereof. This is a significant advancement in thestate of the art.

SUMMARY OF THE INVENTION

The present invention is directed to a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, wherein the salt is selected from a group consisting of monohydrochloride, mono mesylate and mono sulfate. In a certain embodiment,the crystalline salt is substantially pure. One embodiment is directedto the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide. In a certain embodiment, the crystalline mono hydrochloridesalt has an X-ray powder diffraction (XRPD) pattern substantially asillustrated in FIG. 1 after synthesis of the crystalline salt, and, in apreferred embodiment, has characteristic peaks in the XRPD pattern atdiffraction angle 2-theta degrees appearing at least at about 13.4,about 20.5 and about 23.3. In further embodiments, the crystalline monohydrochloride salt has a differential scanning calorimetry (DSC) curvesubstantially as illustrated in FIG. 2 after synthesis, and athermo-gravimetric analysis (TGA) curve substantially as illustrated inFIG. 3 after synthesis. In another embodiment, the crystalline monohydrochloride salt has a DJ-isomer content at 0 days of about 0.1percent peak area (hereinafter referred to as “% peak area”) to about7.0% peak area, as measured by High Performance Liquid Chromatography(HPLC).

Other embodiments of the invention are directed to a crystalline monomesylate salt and a crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide. In a certain embodiment, the crystalline mono mesylate salthas an XRPD pattern substantially as illustrated in FIG. 4 aftersynthesis of the crystalline salt, and, in a preferred embodiment, hascharacteristic peaks in the XRPD pattern at diffraction angle 2-thetadegrees appearing at least at about 9, about 15 and about 23.8. Infurther embodiments, the crystalline mono mesylate salt has a DSC curvesubstantially as illustrated in FIG. 5 after synthesis, and a TGA curvesubstantially as illustrated in FIG. 6 after synthesis.

In a certain embodiment, the crystalline mono sulfate salt has an XRPDpattern substantially as illustrated in FIG. 7 after synthesis of thecrystalline salt, and, in a preferred embodiment, has characteristicpeaks in the XRPD pattern at diffraction angle 2-theta degrees appearingat least at about 15, about 17.8 and about 23.5. In further embodiments,the crystalline mono sulfate salt has a DSC curve substantially asillustrated in FIG. 8 after synthesis, and a TGA curve substantially asillustrated in FIG. 9 after synthesis.

In preferred embodiments, the crystalline mono mesylate salt has aβ-isomer content at 0 days of about 2.0% peak area to about 10.0% peakarea, as measured by HPLC, and the crystalline mono sulfate salt have aβ-isomer content at 0 days of about 3.0% peak area to about 26.0% peakarea, as measured by HPLC.

The present invention is further directed to a pharmaceuticalcomposition comprising a crystalline mono hydrochloride salt,crystalline mono mesylate salt or crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide and a pharmaceutically acceptable excipient. In oneembodiment, the pharmaceutical composition is used for treating acne. Inanother embodiment, the pharmaceutical composition is used for treatingrosacea. In yet another embodiment, the pharmaceutical composition isused for treating a gram positive bacterial infection, wherein the grampositive bacteria is selected from the group consisting ofPropionibacterium acnes, Staphylococcus aureus, Streptococcus pneumonia,Streptococcus pyogenes, and Clostridium difficile.

The present invention is also directed to a method of treating acnecomprising administering to a subject a therapeutically effective amountof a crystalline mono hydrochloride salt, crystalline mono mesylatesalt, or crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

The present invention is also directed to a method of treating rosaceacomprising administering to a subject a therapeutically effective amountof a crystalline mono hydrochloride salt, crystalline mono mesylatesalt, or crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

The present invention is also directed to a method of treating a grampositive bacterial infection, wherein the gram positive bacteria isselected from the group consisting of Propionibacterium acnes.Staphylococcus aureus. Streptococcus pneumonia. Streptococcus pyogenes,and Clostridium difficile, comprising administering to a subject atherapeutically effective amount of a crystalline mono hydrochloridesalt, crystalline mono mesylate salt, or crystalline mono sulfate saltof(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows X-ray powder diffraction (XRPD) analysis of crystallinemono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis and after storage for 7 days at 40° C. and75% relative humidity (RH).

FIG. 2 is a differential scanning calorimetry (DSC) curve of crystallinemono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis.

FIG. 3 is a thermo-gravimetric analysis (TGA) curve of crystalline monohydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis.

FIG. 4 shows XRPD analysis of crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis and after storage for 7 days at 40° C. and75% RH.

FIG. 5 is a DSC curve of crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis.

FIG. 6 is a TGA of crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis.

FIG. 7 shows XRPD analysis of crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis and after storage for 7 days at 40° C. and75% RH.

FIG. 8 is a DSC curve of crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis.

FIG. 9 is a TGA of crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after synthesis.

FIG. 10 shows XRPD analysis of amorphous bis hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

FIG. 11 is a TGA curve and DSC curve overlaid of amorphous bishydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

DETAILED DESCRIPTION OF THE INVENTION Crystalline Salts

Novel crystalline salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide are disclosed herein. After much experimentation anddiscovery, the inventors determined the stable and preferred salt formsof(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, which may be used as a pharmaceutical active ingredient in apharmaceutical composition. The present disclosure teaches how to makethese novel crystalline salts and the superior benefits of them over thefree base of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide and previously known amorphous salts thereof.

Thus, one embodiment of the present invention is a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, wherein the salt is selected from a group consisting of monohydrochloride, mono mesylate and mono sulfate.

In a preferred embodiment, the crystalline salt is substantially pure. Asubstantially pure crystalline salt contains less than about 10% peakarea and, preferably, less than about 4% peak area, total impuritycontent, as measured by HPLC. In a more preferred embodiment, thecrystalline salt is substantially free of an amorphous salt. Preferably,less than about 8% peak area of amorphous salt is present, morepreferably, less than about 5% peak area of amorphous salt is present,and still more preferably, less than about 3% peak area of amorphoussalt is present.

As used herein in reference to the percent peak area of impuritycontent, the term “about” generally means within 10 percent, e.g.,within 5 percent of a given value or range.

The term “crystalline” as used herein refers to compounds in a solidstate having a periodic and repeating three-dimensional internalarrangement of atoms, ions or molecules characteristic of crystals. Theterm crystalline does not necessarily mean that the compound exists ascrystals, but that it has this crystal-like internal structuralarrangement. The term “amorphous” as used herein refers to compoundslacking a crystalline structure: no repeating pattern, only short rangeorder, extensively disordered.

The crystalline salts of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide may be used to treat, prevent, or otherwise amelioratebacterial, viral, parasitic, and fungal infections; cancer (e.g.,prostate, breast, colon, lung melanoma and lymph cancers) and otherdisorders characterized by unwanted cellular proliferation; arthritis;osteoporosis; diabetes; stroke; acute myocardial infarction; aorticaneurysm; neurodegenerative diseases and other conditions for whichtetracycline compounds have been found to be active (see, for example,U.S. Pat. Nos. 5,789,395; 5,834,450; 6,277,061; and 5,532,227, each ofwhich is expressly incorporated herein by reference). In addition, thesalts of the invention can be used to prevent or control importantmammalian and veterinary diseases such as rickettsial infections,sexually transmitted infections, respiratory tract infections, bacterialinfections, ophthalmic infections, anthrax; may serve as therapy inacute intestinal amebiasis, acne, and lyme disease; and may be used forprophylaxis of malaria and the like. Preferably, the crystalline saltsof the present invention may be used to treat bacterial infections andinflammatory skin disorders, which include, without limitation, eczema,dermatitis, psoriasis, pyoderma gangrenosum, acne and rosacea. In oneembodiment, the crystalline salts of the present invention may be usedto treat acne and/or rosacea. For example, the crystalline salts of thepresent invention may be used to treat acne. Nonlimiting examples ofbacterial infections that can be treated by the salts of the inventioninclude infections with gram positive organisms Propionibacterium acnes.Staphylococcus aureus. Streptococcus pneumonia. Streptococcus pyogenes,or Clostridium difficile.

A certain embodiment is the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,1-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

The term “mono hydrochloride salt” as used herein refers to an ioniccompound that results from the neutralization reaction of an acid and abase. The ionic compound (herein, HCl) is composed of a cation and ananion so that the compound is neutral.

General methods for analyzing crystalline salts include crystal analysisby X-ray powder diffraction (XRPD), differential scanning calorimetry(DSC) and thermo-gravimetric analysis (TGA).

XRPD analysis as disclosed herein was collected on a Bruker AXS C2 GADDSdiffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consisted of a single Gbbelmultilayer mirror coupled with a pinhole collimator of 0.3 mm. Thesoftware used for data collection was GADDS for WNT 4.1.16 and the datawas analyzed and presented using Diffrac Plus EVA v 9.0.0.2 or v13.0.0.2. Samples were analyzed under ambient conditions as flat platespecimens using powder as received. Approximately 1-2 mg of the samplewas lightly pressed on a glass slide to obtain a flat surface. Samplesanalyzed under non-ambient conditions were mounted on a silicon waferwith a heat conducting compound. The sample was then heated to theappropriate temperature at approximately 20° C.min⁻¹ and subsequentlyheld isothermally for approximately 1 minute before data collection wasinitiated.

In certain embodiments, the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide has an XRPD pattern substantially as illustrated in FIG. 1after synthesis of the crystalline salt.

The term “XRPD pattern” as used herein refers to the graphicalrepresentation of the data collected by XRPD analysis. XRPD analysis isa technique used to characterize the crystallographic structure, size,and preferred orientation in polycrystalline or powdered solid samples.This diffraction is also used to characterize heterogeneous solidmixtures to determine the percent of crystalline compounds present andcan provide structural information on unknown materials.

The terms “substantially” and “about” as used herein in reference to anXPRD pattern refer to the XPRD pattern wherein a listed peak(s) appearswithin 0.2 degrees 2-theta, including within 0.1 degrees 2-theta of agiven 2-theta value.

In a preferred embodiment, the crystalline mono hydrochloride salt hascharacteristic peaks at diffraction angle 2-theta degrees appearing atleast at about 13.4, about 20.5 and about 23.3, as measured by XRPD. Ina more preferred embodiment, the crystalline mono hydrochloride salt hascharacteristic peaks at diffraction angle 2-theta degrees appearing atleast at about 9.5, about 13.4, about 15.5, about 20.5 and about 23.3,as measured by XRPD, and still more preferable, the crystalline monohydrochloride salt has characteristic peaks at diffraction angle 2-thetadegrees appearing at least at about 9.5, about 13.4, about 15.5, about16.6, about 19.2, about 20.5, about 22.2, and about 23.3.

The term “characteristic peak” as used herein refers to a peak in theXRPD pattern having an intensity at least 20%, more preferably 40%greater than the baseline noise.

TGA and DSC analysis are used to measure thermal behavior and can beused to distinguish between polymorphs. One polymorphic form may exhibitthermal behavior different from that of the amorphous material oranother polymorphic form.

DSC analysis as disclosed herein was collected on a TA Instruments Q2000equipped with a 50 position auto-sampler. The instrument was calibratedfor energy and temperature using certified indium. The calibration forthermal capacity was carried out using sapphire. Typically, 0.5-3.0 mgof each sample, in a pin-holed aluminum pan, was heated at 10° C.min⁻¹from 25° C. to 250° C. A nitrogen purge at 50 ml.min⁻¹ was maintainedover the sample. The instrument control software used was Advantage forQ Series v2.8.0.392 and Thermal Advantage v4.8.3 and the data wasanalyzed using Universal Analysis v4.4A.

DSC is a thermoanalytical technique in which the difference in theamount of heat required to increase the temperature of a sample andreference is measured as a function of temperature. DSC can be used tomeasure a number of characteristic properties of a sample, allowingobservation of crystallization events. Specifically, with DSC, it ispossible to observe small energy changes that occur as mattertransitions from a solid to a liquid crystal and from a liquid crystalto an isotropic liquid. The presence of events in the DSC curve can beused to assess the compound's stability, as well as the presence ofsolvates or hydrates.

TGA is used to determine changes in weight in relation to change intemperature, which may reveal degradation of the compound and thepresence of solvates or hydrates. TGA analysis as disclosed herein wascollected on a TA Instruments Q500 TGA equipped with a 16 positionauto-sampler. The instrument was temperature calibrated using certifiedAlumel and Nickel. Typically, 5-30 mg of each sample was loaded onto apre-weighed platinum crucible and aluminum DSC pan and was heated at 10°C. min⁻¹ from ambient temperature to 300° C. A nitrogen purge at 60ml.min⁻¹ was maintained over the sample. The instrument control and dataanalysis software used was Advantage for Q Series v2.8.0.392 and ThermalAdvantage v4.8.3 and the data was analyzed using Universal Analysisv4.4A.

In a certain embodiment, the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide exhibits a DSC curve substantially as illustrated in FIG. 2.Preferably, the crystalline mono hydrochloride salt analyzed by DSCexhibits no events up to degradation of the crystalline salt.

The term “events” as used herein refers to a change in the sampleassociated with absorption (endothermic) or evolution (exothermic) ofheat causing a change in differential heat flow which is recorded as apeak in the thermogram. Such changes in the sample includedecomposition, degradation, and change of form or morphology, solvate orhydrate. The absence of any events indicates that the compound is stableand is in a low energy form.

The term “substantially,” as used herein in reference to DSC curve meansthe DSC curve demonstrating a peak(s) within 1° C., including within0.5° C. of a given temperature.

In a certain embodiment, the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide exhibits a TGA curve substantially as illustrated in FIG. 3.Preferably, the crystalline mono hydrochloride salt analyzed by TGAexhibits a weight loss of about 1% to about 5% from about 30° C. toabout 200° C. and a weight loss of about 12% to about 16% from about200° C. to about 250° C. and, more preferably, a weight loss of about 3%from about 30° C. to about 200° C. and a weight loss of about 14% toabout 15% from about 200° C. to about 250° C.

The term “substantially,” as used herein in reference to the TGA curvemeans the curve demonstrating a percent weight loss within 1%, includingwithin 0.5% of a given value in relation to temperature change.

In certain embodiments, the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide is stable for at least 4 months, and more preferably for atleast 6 months.

The term “stable” and “stability” as used herein refers to both thephysical form and the chemical purity of the salt. “The salt” as usedherein refers to the disclosed crystalline mono hydrochloride, monomesylate and mono sulfate salts of the present invention.

One measure of the stability of the physical form of the salt ishygroscopicity, which is the propensity of a substance to absorb oradsorb water molecules from the surrounding environment. Whenevermoisture can promote degradation, the salt is stable if it isnon-hygroscopic or mildly hygroscopic above 70% relative humidity (RH).In preferred embodiments, the salt is non-hygroscopic or mildlyhygroscopic above 80% RH, and in more preferred embodiments, the salt isnon-hygroscopic or mildly hygroscopic to 90% RH. “Non-hygroscopic ormildly hygroscopic” as used herein refers to a compound at about 40° C.and at an RH of about 75%, existing over about 80% w/w in solidcrystalline form, preferably over about 90% w/w in solid crystallineform, that absorbs less than 10% w/w water, and preferably, less than 5%w/w water in 8 hours or less. Hygroscopicity (hygroscopic degree) iscalculated based on increase in weight in a compound at comparativepoints of measurement. Another measure of physical stability is thecrystal form of the salt, which may be measured by XPRD.

One measure of chemical purity is defined by the DJ-isomer content ofthe salt. Many tetracyclines are optically active and contain one ormore asymmetric centers. The process by which the asymmetry of such acenter is altered to form the opposite stereochemistry is referred to asepimerization. Tetracyclines undergo reversible epimerization to theless active epi-tetracycline. The rate at which epimerization occurs isdependent on many factors, such as pH, temperature, counter ion, andhumidity. The naturally occurring epimer is typically referred to as a,or the active epimer. The other epimer, known as 1, may or may notpossess biological activity.

(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide has an epimeric center at C₄. The α and β epimers of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide are separable and quantifiable by reversed phase HPLC (HighPerformance Liquid Chromatography) with ultraviolet detector (HPLC-UV)analysis, and measured as percent area under the curve, also referred toas percent peak area. Although the epimer of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide is believed to be non-toxic, under certain conditions it maylack the anti-bacterial efficacy of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide and, therefore, is considered an undesirable degradationproduct.

The lower the β-isomer content, the higher the chemical purity of thesalt. The β-isomer content is measured after synthesis of the salt andcompared with the measured β-isomer content after storage for adesignated period of time. Where the β-isomer content does notsignificantly increase after storage, there has been no negative effectof storage on the chemical purity of the salt and the salt is stable forthat designated period of time. Since moisture uptake by thetetracycline may be a contributing factor to epimerization, a salt formof(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide that is not significantly hygroscopic may provide an innateresistance to epimerization caused by humidity.

Another measure of chemical purity is defined by the content of otherrelated impurities, by-products or degradation products of the salt,which represents the morphology of the compound. The lower the contentof total impurities as measured by HPLC, generally, by HPLC-UV, thehigher the chemical purity of the salt. The total impurity content ismeasured after synthesis of the salt and compared with the measuredtotal impurity content after storage for a designated period of time.Where the total impurity content does not significantly increase afterstorage, there has been no negative effect of storage on the chemicalpurity of the salt and the salt is stable for that designated period oftime.

In certain embodiments, at 0 days, the crystalline mono hydrochloridesalt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide has a total impurity content of less than about 8% and,preferably, less than about 4%. In another embodiment, the crystallinemono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide has a total impurity content after storage for about 6 monthsat about 40° C. and about 75% RH of less than about 10% and, preferably,less than about 6%. In a certain embodiment, the salt has a totalimpurity content after storage for about 6 months at about 40° C. andabout 75% RH not more than about 80% peak area greater than the totalimpurity content at about 0 days, and preferably, not more than about50% peak area greater than the total impurity content at about 0 days.

In a certain embodiment of the present invention, the crystalline monohydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide is stable and has a β-isomer content after storage for about75 days at about 40° C. and about 75% RH not more than about 20% peakarea greater than the 3-isomer content at about 0 days. In a preferredembodiment, the salt has a 3-isomer content after storage for about 75days at about 40° C. and about 75% RH not more than about 10% peak areagreater than the β-isomer content at about 0 days; in a more preferredembodiment, the β-isomer content after storage for about 75 days atabout 40° C. and about 75% RH is not more than about 1% peak areagreater than the β-isomer content at about 0 days; and in a furtherpreferred embodiment, the β-isomer content after storage for about 75days at about 40° C. and about 75% RH is about equal to the β-isomercontent at about 0 days. “After synthesis” as used herein refers to lessthan about one day from the time of confirmation of synthesis, alsoreferred to as “0 days.”

In a certain embodiment, the β-isomer content of the crystalline monohydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide at 0 days is about 0.1% to about 7.0% peak area, preferablyabout 1.0% to about 6.0% peak area, more preferably about 2.0% to about4.0% peak area, and most preferably about 3.0% to about 4.0% peak area.In a certain embodiment, the β-isomer content of the crystalline monohydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide after storage for at least 3 months is about 2.0% to about8.0% peak area, and more preferably about 3.0% to about 4.0% peak area.In other embodiments, the β-isomer content after storage for at least 6months is about 0.1% to about 10.0% peak area, and more preferably about2.0% to about 8.0% peak area. In a certain embodiment, the β-isomercontent of the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide at 0 days is less than about 6.0% peak area and after storagefor about 75 days at ambient conditions is less than about 6.0% peakarea. Preferably, the β-isomer content at 0 days is less than about 4.5%peak area and after storage for about 75 days at ambient conditions isless than about 4.5% peak area, and in a still further embodiment, theβ-isomer content at 0 days is about 3.8% peak area and after storage forabout 75 days at ambient conditions is about 3.8% peak area.

Ambient conditions, as used herein, means a temperature of about 20° C.to about 25° C. and an RH of about 40%.

Another embodiment of the present invention is the crystalline monomesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide.

The term “mono mesylate salt” as used herein refers to an ionic compoundthat results from the neutralization reaction of an acid and a base. Thecompound is composed of a cation and an anion (herein, CH₃SO₂) so thatthe compound is neutral.

In certain embodiments, the crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide has an XRPD pattern substantially as illustrated in FIG. 4after synthesis of the crystalline salt.

In a preferred embodiment, the crystalline mono mesylate salt hascharacteristic peaks at least appearing at diffraction angle 2-thetadegrees appearing at about 9, about 15 and about 23.8, as measured byXRPD. In a more preferred embodiment, the crystalline mono mesylate salthas characteristic peaks at diffraction angle 2-theta degrees appearingat least at about 9, about 15, about 22.7 and about 23.8, as measured byXRPD, and still more preferable, the crystalline mono mesylate salt hascharacteristic peaks at diffraction angle 2-theta degrees appearing atleast at about 9, about 15, about 22, about 22.7 and about 23.8.

In a certain embodiment, the crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide exhibits a DSC curve substantially as illustrated in FIG. 5.Preferably, the crystalline mono mesylate salt analyzed by DSC exhibitsno events up to degradation of the crystalline salt.

In a certain embodiment, the crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide exhibits a TGA curve substantially as illustrated in FIG. 6.Preferably, the crystalline mono mesylate salt analyzed by TGA exhibitsa weight loss of about 1% to about 4% from about 30° C. to about 200° C.and a weight loss of about 3% to about 10% from about 200° C. to about250° C. and, more preferably, a weight loss of about 2% to about 3% fromabout 30° C. to about 200° C. and a weight loss of about 6% to about 7%from about 200° C. to about 250° C.

In a certain embodiment of the present invention, the crystalline monomesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-carboxylicacid amide is stable and has a β-isomer content after storage for about75 days at about 40° C. and about 75% RH not more than about 20% peakarea greater than the β-isomer content at about 0 days. In a preferredembodiment, the salt has a β-isomer content after storage for about 75days at about 40° C. and about 75% RH not more than about 10% peak areagreater than the β-isomer content at about 0 days; in a more preferredembodiment, the 0-isomer content after storage for about 75 days atabout 40° C. and about 75% RH is not more than about 1% peak areagreater than the β-isomer content at about 0 days; and in a furtherpreferred embodiment, the β-isomer content after storage for about 75days at about 40° C. and about 75% RH is about equal to the 3-isomercontent at about 0 days.

In a certain embodiment, the β-isomer content of the crystalline monomesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-carboxylicacid amide at 0 days is about 2.0% to about 10.0% peak area, preferablyabout 2.0% to about 6.0% peak area, and more preferably about 2.0% toabout 3.0% peak area.

Another embodiment of the present invention is a crystalline monosulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,1,12a-octahydro-naphthacene-2-carboxylicacid amide.

The term “mono sulfate salt” as used herein refers to an ionic compoundthat results from the neutralization reaction of an acid and a base. Thecompound is composed of a cation and an anion (herein, SO₄₂) so that thecompound is neutral.

In certain embodiments, the crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide has an XRPD pattern substantially as illustrated in FIG. 7after synthesis of the crystalline salt.

In a preferred embodiment, the crystalline mono sulfate salt hascharacteristic peaks at diffraction angle 2-theta degrees appearing atleast at about 15, about 17.8 and about 23.5, as measured by XRPD. In amore preferred embodiment, the crystalline mono sulfate salt hascharacteristic peaks at diffraction angle 2-theta degrees appearing atleast at about 15, about 17.8, about 22.5 and about 23.5, as measured byXRPD. In a still more preferred embodiment, the crystalline mono sulfatesalt has characteristic peaks at diffraction angle 2-theta degreesappearing at least at about 15, about 17.8, about 19.0, about 22.5 andabout 23.5, as measured by XRPD.

In a certain embodiment, the crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide exhibits a DSC curve substantially as illustrated in FIG. 8.Preferably, the crystalline mono sulfate salt analyzed by DSC exhibitsno events up to degradation of the crystalline salt.

In a certain embodiment, the crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide exhibits a TGA curve substantially as illustrated in FIG. 9.Preferably, the crystalline mono sulfate salt analyzed by TGA exhibits aweight loss of about 1% to about 5% from about 30° C. to about 200° C.and a weight loss of about 12% to about 16% from about 200° C. to about250° C. and, more preferably, a weight loss of about 3% to about 4% fromabout 30° C. to about 200° C. and a weight loss of about 13% to about14% from about 200° C. to about 250° C.

In a certain embodiment of the present invention, the crystalline monosulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide is stable and has a β-isomer content after storage for about75 days at about 40° C. and about 75% RH not more than about 20% peakarea greater than the β-isomer content at about 0 days. In a preferredembodiment, the salt has a β-isomer content after storage for about 75days at about 40° C. and about 75% RH not more than about 10% peak areagreater than the β-isomer content at about 0 days; in a more preferredembodiment, the 0-isomer content after storage for about 75 days atabout 40° C. and about 75% RH is not more than about 1% peak areagreater than the β-isomer content at about 0 days; and in a furtherpreferred embodiment, the β-isomer content after storage for about 75days at about 40° C. and about 75% RH is about equal to the β-isomercontent at about 0 days.

In a certain embodiment, the β-isomer content of the crystalline monosulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide at 0 days is about 3.0% to about 26.0% peak area, preferablyabout 5.0% to about 20.0% peak area, and most preferably about 6.0% toabout 10.0% peak area.

Pharmaceutical Compositions

One embodiment of the invention is directed to a pharmaceuticalcomposition comprising a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, wherein the salt is selected from a group consisting of monohydrochloride, mono mesylate, and mono sulfate, and a pharmaceuticallyacceptable excipient.

The pharmaceutical composition of the present invention comprises aneffective amount of a crystalline salt, a pharmaceutically acceptableexcipient, and, in some embodiments, it may also contain one or moreadditional active ingredients. The content of crystalline salt in thepharmaceutical composition of the present invention varies depending onthe subject of administration, route of administration and targetdisease, among other variables. The pharmaceutical composition of thepresent invention may be administered orally, topically (e.g.,transdermal, etc.), vaginally, rectally, or parenterally (e.g.,intravenous, etc.). Preferably, the pharmaceutical composition of thepresent invention may be used for treating bacterial infections andinflammatory skin disorders. For example, the pharmaceutical compositionof the present invention may be used for treating acne and/or rosacea,e.g., for treating acne, or for treating infections with gram positivebacteria, wherein the gram positive bacteria is selected from the groupconsisting of Propionibacterium acnes. Staphylococcus aureus.Streptococcus pneumonia. Streptococcus pyogenes, and Clostridiumdifficile.

Examples of topical administration of the pharmaceutical compositioninclude transdermal, buccal or sublingual application. For topicalapplications, the pharmaceutical composition can be suitably admixed ina pharmacologically inert topical carrier, such as a gel, an ointment, alotion or a cream. Such pharmacologically inert topical carriers includewater, glycerol, alcohol, propylene glycol, fatty alcohols,triglycerides, fatty acid esters, or mineral oils. Other possiblepharmacologically inert topical carriers are liquid petrolatum,isopropylpalmitate, polyethylene glycol, ethanol 95/%, polyoxyethylenemonolauriate 5% in water, sodium lauryl sulfate 5% in water, and thelike. In addition, materials such as anti-oxidants, humectants,viscosity stabilizers and the like also may be added.

For oral administration, the crystalline salt of the present inventionmay be administered as a capsule, tablet or granule. Tablets may containvarious excipients such as microcrystalline cellulose, sodium citrate,calcium carbonate, dicalcium phosphate and glycine, along with variousdisintegrants such as starch (and preferably corn, potato or tapiocastarch), alginic acid and certain complex silicates, together withgranulation binders like polyvinylpyrrolidone, sucrose, gelatin andacacia. In a certain embodiment, the tablet may be film coated.Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate and talc are often very useful for tablets. Other solidcompositions may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the crystalline salt may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents, together with such diluents aswater, ethanol, propylene glycol, glycerin and various like combinationsthereof. The pharmaceutical compositions of the invention may beformulated such that the crystalline salt is released over a period oftime after administration.

Preparing such pharmaceutical compositions of the crystalline salt ofthe present invention along with a pharmaceutically acceptable excipientand, optionally, an additional active ingredient, may be done by anyconventional technique known in the art.

In an embodiment, the crystalline salt present in the pharmaceuticalcomposition is about 0.01% to about 90% by weight relative to the wholecomposition. A suitable therapeutically effective amount of thecrystalline salt will typically range from about 0.01 mg/kg to about 1g/kg of body weight per day; in another embodiment, from about 1 mg/kgto about 600 mg/kg body weight per day; in another embodiment, fromabout 1 mg/kg to about 250 mg/kg body weight per day; in anotherembodiment, from about 10 mg/kg to about 400 mg/kg body weight per day;in another embodiment, from about 10 mg/kg to about 200 mg/kg of bodyweight per day; in another embodiment, from about 10 mg/kg to about 100mg/kg of body weight per day; in one embodiment, from about 10 mg/kg toabout 25 mg/kg body weight per day; in another embodiment, from about 1mg/kg to about 10 mg/kg body weight per day; in another embodiment, fromabout 0.001 mg/kg to about 100 mg/kg of body weight per day; in anotherembodiment, from about 0.001 mg/kg to about 10 mg/kg of body weight perday; and in another embodiment, from about 0.001 mg/kg to about 1 mg/kgof body weight per day. In a certain embodiment, when a pharmaceuticalcomposition described herein is administered orally, a suitabletherapeutically effective amount of the crystalline salt is about 0.01to about 100 milligrams per kilogram of body weight of recipient perday, preferably about 0.1 to about 50 milligrams per kilogram bodyweight of recipient per day, more preferably from about 0.1 to about 20milligrams per kilogram body weight of recipient per day, and even morepreferably from about 0.1 to about 10 milligrams per kilogram bodyweight of recipient per day. The desired dose may be administered oncedaily, or by several sub-divided doses, e.g., 2 to 5 sub-divided doses,at appropriate intervals through the day, or other appropriate schedule.

The term “pharmaceutically acceptable excipient” as used hereinincludes, but is not limited to, one of more of the following: polymers,resins, plasticizers, fillers, lubricants, diluents, binders,disintegrants, solvents, co-solvents, surfactants, buffer systems,preservatives, sweetener agents, flavoring agents, pharmaceutical-gradedyes or pigments, chelating agents, viscosity agents, and combinationsthereof. Pharmaceutically acceptable excipients can be used in anycomponent in making the dosage form, i.e. core tablet or coating.Flavoring agents and dyes and pigments among those useful herein includebut are not limited to those described in Handbook of PharmaceuticalExcipients (4th Ed., Pharmaceutical Press 2003). Suitable co-solventsinclude, but are not limited to, ethanol, isopropanol, acetone, andcombinations thereof. Suitable surfactants include, but are not limitedto, polyoxyethylene sorbitan fatty acid esters, polyoxyethylenemonoalkyl ethers, sucrose monoesters, simethicone emulsion, sodiumlauryl sulfate, Tween 80®, and lanolin esters, ethers, and combinationsthereof. Suitable preservatives include, but are not limited to, phenol,alkyl esters of parahydroxybenzoic acid, benzoic acid and the saltsthereof, boric acid and the salts thereof, sorbic acid and the saltsthereof, chlorbutanol, benzyl alcohol, thimerosal, phenylmercuricacetate and nitrate, nitromersol, benzalkonium chloride, cetylpyridiniumchloride, methyl paraben, propyl paraben, and combinations thereof.Suitable fillers include, but are not limited to, starch, lactose,sucrose, maltodextrin, and microcrystalline cellulose. Suitableplasticizers include, but are not limited to, triethyl citrate,polyethylene glycol, propylene glycol, dibutyl phthalate, castor oil,acetylated monoglycerides, triacetin, and combinations thereof. Suitablepolymers include, but are not limited to, ethylcellulose, celluloseacetate trimellitate, hydroxypropylmethylcellulose phthalate, celluloseacetate phthalate, polyvinyl acetate phthalate, and Eudragit® L 30-D,Eudragit® L 100-55, Eudragit® F530D and Eudragit® S 100 (Rohm PharmaGmbH and Co. KG, Darmstadt, Germany), Acryl-EZE® and Sureteric®(Colorcon, Inc., West Point, Pa.), and combinations thereof. Suitablelubricants include, but are not limited to, magnesium stearate, stearicacid, talc, and combinations thereof.

The term “additional active ingredient” as used herein includes anyagent known in the art to treat, prevent or reduce the symptoms of thecondition being treated by the pharmaceutical composition. Such agents,include but are not limited to agents known to treat, prevent or reducethe symptoms of bacterial infections and inflammatory skin disorders.

The improved stability of the crystalline salts of the present inventionmeans that the crystals are less hygroscopic, i.e., less sensitive tohumidity, so that a pharmaceutical composition containing thecrystalline salt can be stored for a longer period of time thanpreviously known pharmaceutical compositions.

In a certain embodiment, the pharmaceutical composition comprises themono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide and a pharmaceutically acceptable excipient. In anotherembodiment, the pharmaceutical composition comprises the mono mesylatesalt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide and a pharmaceutically acceptable excipient. In a stillfurther embodiment, the pharmaceutical composition comprises the monosulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide and a pharmaceutically acceptable excipient.

In a certain embodiment, the invention is directed to a pharmaceuticalcomposition comprising(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable excipient for use in treating a bacterialinfection, e.g., a Streptococcus pyogenes and Clostridium difficilebacterial infection. In a preferred embodiment, the pharmaceuticalcomposition comprises a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide selected from the group consisting of mono hydrochloride,mono mesylate and mono sulfate salt.

The phrase “pharmaceutically acceptable salt” of a compound as usedherein means a salt that is pharmaceutically acceptable and thatpossesses the desired pharmacological activity of the parent compound.Pharmaceutically acceptable salts include salts of acidic or basicgroups present in a compound of the invention. Pharmaceuticallyacceptable acid addition salts include, but are not limited to,hydrochloride, hydrobromide, hydroiodide, nitrate, mesylate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Suitable base saltsinclude, but are not limited to, aluminum, calcium, lithium, magnesium,potassium, sodium, zinc, and diethanolamine salts. Preferably, thepharmaceutically acceptable salt is a crystalline salt. Even morepreferably, the pharmaceutically acceptable salt is a crystalline saltselected from mono hydrochloride, mono mesylate, and mono sulfate.

Methods of Use

One embodiment of the invention is directed to a method for treatingacne and/or rosacea comprising administering to a subject atherapeutically effective amount of a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, wherein the crystalline salt is selected from a groupconsisting of mono hydrochloride, mono mesylate and mono sulfate. In oneembodiment, the invention is directed to a method of treating acne. Inanother embodiment, the invention is directed to a method of treatingrosacea.

The term “treating” as used herein includes therapeutic and/orprophylactic treatment of acne and/or rosacea or other conditionsdescribed herein. The treatment includes the diminishment or alleviationof at least one symptom associated with acne and/or rosacea or at leastone symptom associated with another condition described herein.

The term “therapeutically effective amount” as used herein means anamount of a compound or composition high enough to significantlypositively modify the symptoms and/or condition to be treated, but lowenough to avoid serious side effects (at a reasonable risk/benefitratio), within the scope of sound medical judgment. The therapeuticallyeffective amount of active ingredient for use in the method of theinvention herein will vary with the particular condition being treated,the age and physical condition of the patient to be treated, theseverity of the condition, the duration of the treatment, the nature ofconcurrent therapy, the particular active ingredient being employed, theparticular pharmaceutically-acceptable excipients utilized, and likefactors within the knowledge and expertise of a skilled physician orveterinarian. Various suitable therapeutically effective amounts aredescribed above.

The term “subject” as used herein is an animal. “Subject” includes,without limitation, a human, mouse, rat, guinea pig, dog, cat, horse,cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. In oneembodiment, “subject” is a mammal. In another embodiment, “subject” is ahuman.

A certain embodiment is directed to the method for treating acnecomprising administering to a subject a therapeutically effective amountof the crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide. In an embodiment thereof, the amount of the monohydrochloride salt employed is between about 10 mg and about 2000 mg,and preferably between about 25 mg and about 500 mg. In a certainembodiment, the mono hydrochloride salt is administered at least oncemonthly, preferably, weekly, more preferably, bi-weekly, and mostpreferably, the mono hydrochloride salt is administered daily.

Another embodiment is directed to the method for treating acnecomprising administering to a subject a therapeutically effective amountof the crystalline mono mesylate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide. In certain embodiments thereof, the amount of crystallinemono mesylate salt employed is between about 10 mg and about 2000 mg,and preferably between about 25 mg and about 500 mg. In a certainembodiment, the mono mesylate salt is administered at least oncemonthly, preferably, weekly, more preferably, bi-weekly, and mostpreferably, the mono mesylate salt is administered daily.

A further embodiment is directed to the method for treating acnecomprising administering to a subject a therapeutically effective amountof the crystalline mono sulfate salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide. In certain embodiments, the amount of crystalline monosulfate salt employed is between about 10 mg and about 2000 mg, andpreferably between about 25 mg and about 500 mg. In a certainembodiment, the mono sulfate salt is administered at least once monthly,preferably, weekly, more preferably, bi-weekly, and most preferably, themono sulfate salt is administered daily.

Yet another embodiment of the invention is directed to a method oftreating a gram positive bacterial infection comprising administering toa subject a therapeutically effective amount of a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, wherein the crystalline salt is selected from a groupconsisting of mono hydrochloride, mono mesylate and mono sulfate. Grampositive bacterial infections include Propionibacterium acnes.Staphylococcus aureus. Streptococcus pneumonia. Streptococcus pyogenes,and Clostridium diflicile infections.

An additional embodiment of the invention is directed to a method oftreating a bacterial infection, e.g., a gram positive bacterialinfection selected from Streptococcus pyogenes and Clostndium difficileinfection, comprising administering to a subject a therapeuticallyeffective amount of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide or pharmaceutically acceptable salt thereof. In a preferredembodiment, the method comprises administering to a subject atherapeutically effective amount of a crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, preferably the salt is mono hydrochloride, mono mesylate ormono sulfate salt.

The following examples will illustrate the practice of the presentinvention in some of the preferred embodiments. Other embodiments withinthe scope of the claims will be apparent to one skilled in the art.

EXAMPLES

The following examples illustrate the synthesis of the compoundsdescribed herein.

Synthesis of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (“the Free Base”)

A solution of 7-formylsancycline TFA salt (2.23 g) andN,O-dimethylhydroxylamine hydrochloride (780 mg) inN,N-dimethylacetamide (15 mL) was stirred for 10 minutes at roomtemperature under argon atmosphere. To this solution was added sodiumcyanoborohydride (302 mg). The solution was stirred for 5 minutes andmonitored by LC-MS. The reaction mixture was poured into diethyl ether,and the resulting precipitates were collected by filtration undervacuum. The crude product was purified by prep-HPLC using a C18 column(linear gradient 10-40% acetonitrile in 20 mM aqueous triethanolamine,pH 7.4). The prep-HPLC fractions were collected, and the organic solvent(acetonitrile) was evaporated under reduced pressure. The resultingaqueous solution was loaded onto a clean PDVB SPE column, washed withdistilled water, then with a 0.1 M sodium acetate solution followed bydistilled water. The product was eluted with acetonitrile. The eluentwas concentrated under reduced pressure, 385 mg was obtained as freebase.

Synthesis of Crystalline Mono Hydrochloride Salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (the “Crystalline Mono Hydrochloride Salt”)

Crude(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide (100 g, app. 35% assay) was purified on preparative columnchromatography. The desired fractions (8-10 liters) were combined andthe pH was adjusted to 7.0-7.5 using ammonium hydroxide. This aqueoussolution was extracted 3 times with dichloromethane (4 liters eachtime). The dichloromethane layers were combined and concentrated underreduced pressure. The residue was suspended in ethanol (800 ml) and 20ml water was added. The pH was gradually adjusted to pH 1.6-1.3 using1.25M hydrochloric acid in methanol and the mixture was stirred for20-60 minutes at which point the free base was completely dissolved. Thesolution was concentrated under reduced pressure to 200-250 ml and wasseeded with(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide mono HCl crystals (100-200 mg). The stirring was continuedfor 2-18 hours while the slurry was kept at ≤5° C. The resultingcrystals were filtered, washed with ethanol (50 mL) and dried underreduced pressure to a constant weight. 20 g crystalline(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide mono hydrochloride was isolated in ≥90% purity and ≥90%assay.

Synthesis of Crystalline Mono Mesylate Salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid (the “Crystalline Mesylate Salt”)

(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide free base (74 mg) was suspended in ethanol (740 μl) andheated with stirring to 60° C. (bath temperature). Methane sulfonic acid(1.1 eq, 167 μl as 1M solution in THF) was added and most of the soliddissolved. After five minutes, the suspension was cooled to ambienttemperature over approximately 1.75 hours (uncontrolled in oil bath). By53° C., solid had precipitated which was filtered at ambient temperatureunder reduced pressure. A further portion of ethanol (200 μl) was addedto aid filtration, as the suspension was viscous. The cake was washedwith n-hexane (400 μl) and air dried on filter for approximately 30minutes to yield 59 mg (67% yield) of yellow solid.

Synthesis of Crystalline Mono Sulfate Salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid (the “Crystalline Sulfate Salt”)

(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide free base (86 mg) was suspended in ethanol (500 μl) andheated with stirring to 63° C. (bath temperature) at which temperaturemost of the free base had dissolved. Sulfuric acid (1.1 eq, 1941l as 1Msolution in water) was added and all of the solid dissolved. Thesolution was cooled to ambient temperature over approximately 1.75 hours(uncontrolled in oil bath) at which temperature no solid hadprecipitated. Methyl t-butyl ether (MtBE) was added as an antisolvent(4×50 μl). Each addition caused a cloud point, but the solidre-dissolved on stirring. The solution was stirred with a stopper forapproximately 3 hours after which time solid precipitated. The solid wasfiltered under reduced pressure and washed with MtBE (3×200 μl) and airdried on filter for approximately 45 minutes to yield 93 mg (90% yield)of yellow solid.

Comparative Example 1 Synthesis of Amorphous Bis Hydrochloride Salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide

(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide free base (1 g) was suspended in methanol (50 mL). Thefreebase was converted to the hydrochloride salt by adding an excess ofmethanolic HCl followed by under reduced pressure evaporation to give1.1 g yellow solid: MS (Mz+1=488). 1H NMR (300 MHz, CD3OD) δ 7.46 (d,1H, J=8.6 Hz), 6.81 (d, 1H, J=8.6 Hz), 4.09 (d, 1H, J=1.0 Hz), 3.79 (d,1H, J=13.1 Hz), 3.73 (d, 1H, J=13.1 Hz), 3.36 (m, 1H), 3.27 (s, 3H),3.08-2.95 (8H), 2.61 (s, 3H), 2.38 (t, 1H, J=14.8), 2.22 (m, 1H), 1.64(m, 1H). An XRPD pattern is shown in FIG. 10 and a TGA and DSC curveoverlaid are shown in FIG. 11.

Comparative Example 2 Synthesis of Amorphous Mono Hydrochloride Salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide

A sample of Crystalline Mono Hydrochloride Salt (2.09 g) was dissolvedin water (250 ml, 120 vols), filtered and frozen in a −78° C. bath.Water was removed from the solidified sample using a lyophilizer for 110hours to yield the amorphous mono hydrochloride salt as a fluffy yellowsolid, that was confirmed to be amorphous by XRPD analysis

Testing

In order to determine the stability of Crystalline Mono HydrochlorideSalt, the β-isomer content of the salt was determined by HPLC-UVanalysis and compared to the β-isomer content calculated after the saltwas stored in an amber glass vial for approximately 75 days at ambientconditions. The results are shown in Table 1 below. As evidenced by thedata collected, the β-isomer content did not increase over time andtherefore, storage did not negatively affect the chemical purity of theCrystalline Mono Hydrochloride Salt.

TABLE 1 Formation of β-isomer in the Crystalline Mono Hydrochloride Saltafter Storage. Chemical Purity Chemical Purity Sample at 0 days at 75days Crystalline Mono 93.8% (3.8% β-isomer) 94.8% (3.8% β-isomer)Hydrochloride Salt

Other samples of the Crystalline Mono Hydrochloride Salt were analyzedby HPLC-UV and due to minor variations in the synthetic process, purityat 0 days was found to be 93.8% (3.5% β-isomer) and 95.8% (3.4%β-isomer). The chemical purity for these samples after storage was nottested.

Further evidence of stability was demonstrated by analysis of the totalimpurity content of Crystalline Mono Hydrochloride Salt by HPLC-UV at 0days and again after storage for about 6 months at about 40° C. andabout 75% RH and was found to be 4% peak area and 6% peak area,respectively.

In tests conducted on the stability of Comparative Example 1, after just48 hours of storage at about 40° C. and about 75% RH, the β-isomercontent increased to 31.7% peak area from 3.6% peak area at 0 days.After storage for 36 days at about 25° C. and about 60% RH, the β-isomercontent was calculated as 8.7% peak area. In another test, the compoundof Comparative Example 1 had a total impurity content of 4.2% peak areaat 0 days and 34.6% peak area after storage for about 2 days at about40° C. and about 75% RH. Accordingly, Comparative Example 1 exhibited amuch higher increase in total impurity content and was significantlyless stable than Crystalline Mono Hydrochloride Salt.

The β-isomer content of Comparative Example 2 was also determined byHPLC-UV analysis at 0 days and compared to the β-isomer contentcalculated after the compound was stored in a clear glass vial forapproximately 75 days at ambient conditions. The results are shown inTable 2 below. As evidenced by the data, after storage for 75 days, theβ-isomer content increased by over 80% and, therefore, the storagenegatively affected the chemical purity of Comparative Example 2.

TABLE 2 Formation of β-isomer in Comparative Example 2 after Storage.Chemical Purity Chemical Purity Sample at 0 days at 75 days Comparative94.3% (4.4% β-isomer) 90.4% (7.8% β-isomer) Example 2

The stability of Crystalline Mono Hydrochloride Salt was compared to theamorphous mono hydrochloride salt of Comparative Example 2. The resultsof various tests demonstrating advantages and disadvantages of theCrystalline Mono Hydrochloride Salt and Comparative Example 2 are shownin Table 3 below.

TABLE 3 Advantages and Disadvantages of the Crystalline MonoHydrochloride Salt and Comparative Example 2. Solid form AdvantagesDisadvantages Crystalline Non-hygroscopic to 90% RH Some loss ofcrystallinity upon Mono pressing and milling Hydrochloride No change inform or β-isomer — Salt content upon milling or pressing No increase inβ-isomer content — upon storage under ambient conditions ComparativeHigh glass transition (166° C.) Hygroscopic above 70% RH Example 2Stable to crystallization upon Change in form (from amorphous storageand heat/cool cycle to crystalline) to Crystalline Mono HydrochlorideSalt observed above 75% RH — Increase in β-isomer content upon storageat 63% RH — Increase in β-isomer content upon pressing and milling —Increase in β-isomer content upon storage under ambient conditions —Faster rate of β-isomer formation upon exposure to solvent as comparedto Crystalline Mono Hydrochloride Salt

HPLC-UV and XRPD analysis were conducted on samples of Crystalline MonoHydrochloride Salt, Crystalline Mesylate Salt and Crystalline SulfateSalt at 0 days and after storage for 7 days at 40° C. and 75% RH. FIGS.1, 4 and 7 show the XRPD analysis of the Crystalline Mono HydrochlorideSalt, Crystalline Mesylate Salt and Crystalline Sulfate Salt at 0 daysand after storage at 40° C. and 75% RH with the graphs overlaid forcomparison. As shown in FIG. 1, Crystalline Mono Hydrochloride Saltshowed no change in crystal form after storage. As shown in FIG. 4,Crystalline Mesylate Salt also showed no change in crystal form afterstorage. The changes shown in the figure are in intensity and resolutionrather than in peak position, which, if present, would indicate changein crystal form. Accordingly, Crystalline Mono Hydrochloride Salt andCrystalline Mesylate Salt are physically stable, as shown by FIGS. 1 and4. As shown in FIG. 7, Crystalline Sulfate Salt showed increasedcrystalline content after storage.

The results of HPLC-UV analysis for the same samples of CrystallineMesylate Salt and Crystalline Sulfate Salt are shown in Table 4. As theβ-isomer content did not increase after storage, these salts were notnegatively affected by storage.

TABLE 4 Formation of β-isomer in Crystalline Mesylate Salt andCrystalline Sulfate Salt after Storage. Chemical Purity Chemical Purityat 7 days, Sample at 0 days 40° C. and 75% RH Crystalline Mesylate Salt92% (3% β-isomer) 98% (2% β-isomer) Crystalline Sulfate Salt 88% (9%β-isomer) 91% (9% β-isomer)

In addition, differential scanning calorimetry (DSC) andthermo-gravimetric (TGA) analysis of Crystalline Mono HydrochlorideSalt, Crystalline Mesylate Salt and Crystalline Sulfate Salt aftersynthesis was conducted. The DSC curves are shown in FIGS. 2, 5 and 8and the TGA curves are shown in FIGS. 3, 6 and 9. These figures showthat, by DSC analysis, there are no events up to degradation of thesalts, thereby, confirming stability of the salts at raisedtemperatures. The TGA curves show that no hydrates or solvates werepresent. The observed apparent weight loss is due to instability of themachine.

Table 5 below compares the DSC and TGA analysis of the crystalline saltsof the present invention with the crystalline free base and ComparativeExample 1.

TABLE 5 DSC and TGA Analysis of Crystalline Mono Hydrochloride Salt,Crystalline Mesylate Salt and Crystalline Sulfate Salt, Crystalline FreeBase and Comparative Example 1. Differential Scanning Sample CalorimetryThermo-Gravimetric Analysis Crystalline Mono No events up to 3% weightloss from about 30° C. to Hydrochloride Salt degradation about 200° C.14% weight loss from about 200° C. to about 250° C. Crystalline Noevents up to 3% weight loss from about 30° C. to Mesylate Saltdegradation about 200° C. 7% weight loss from about 200° C. to about250° C. Crystalline Sulfate No events up to 3% weight loss from about30° C. to Salt degradation about 200° C. 14% weight loss from about 200°C. to about 250° C. Crystalline Free Endothermic peak at 15% weight lossfrom about 30° C. Base 175° C. (ΔH 72 J · g⁻¹) to decomposition at lessthan 220° C. Comparative Broad endothermic 7% weight loss from about 30°C. to Example 1 peak between about about 106° C. 20° C. and about 8%weight loss from about 106° C. 200° C. to about 200° C. 23% weight lossfrom about 200° C. to about 300° C.

Accordingly, Crystalline Mono Hydrochloride Salt, Crystalline MesylateSalt and Crystalline Sulfate Salt are more stable than crystalline freebase and Comparative Example 1.

Antimicrobial Activity

Antimicrobial activity of the Crystalline Mono Hydrochloride Salt wasassessed according to anti-anaerobic activity, mechanism of action andin vivo efficacy studies as detailed herein. Whether either amorphousbis hydrochloride salt or crystalline mono hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide is used to prepare samples for these studies, it is wellunderstood in the art that efficacy data would be the same for all saltforms, since the compound is placed into solution prior to testing.Accordingly, whether starting with the Crystalline Mono HydrochlorideSalt or amorphous bis hydrochloride salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, the free base (as defined above; herein “the active”) iswhat is being tested.

For the studies below, samples were prepared with bis hydrochloridesalt, and the data is expressed based on the free base (“active”). Theoverall anti-anaerobic microbial activity of the Crystalline MonoHydrochloride Salt can be seen via in vitro study of the active against37 representative strains of anaerobic bacteria and the results compiledin Table 6. The active demonstrated relatively potent activity (i.e.,minimum inhibitory concentration (MIC) of 4 g/mL or less) against manyspecies of Gram-positive bacteria, including P. acnes. Overall, theactivity of the active was similar to that of tetracycline anddoxycycline but less than that of minocycline. Organisms with high MICvalues for the active (MIC≥16 μg/mL) included C. perfringens and S.constellatus.

MIC values for the Gram-negative anaerobes are shown in Table 6. Thetetracycline-resistant strains were cross-resistant to the active. Theactive and the other tetracyclines demonstrated potent activity againstE. corrodens and Fusobacterium spp., moderate activity against P.melaninogenica (1 of 2 strains) and V. parvula, and poor activityagainst P. asaccharolytica.

TABLE 6 Summary of in vitro MIC testing of the Active Against AnaerobicGram-Positive and Gram-Negative Bacteria. The Active TET DOX MINOrganism/ ATCC MIC MIC MIC MIC Micromyx No. No. (μg/mL) (μg/mL) (μg/mL)(μg/mL) Bifidobacterium bifidum 15696 1 1 0.5 0.25 3965 Bifidobacteriumbrevi 15698 1 1 0.5 0.25 3967 Bifidobacterium infantis 15702 0.5 1 0.50.25 3966 Bifidobacterium longum 15707 4 2 1 1 3968 Clostridiumperfringens — 16 >16 16 16 3414 Clostridium perfringens — 16 >16 16 83518 Clostridium difficile — 0.12 0.5 0.06 0.03 3579 Clostridiumdifficile — 0.12 0.5 0.06 0.03 3584 Lactobacillus — 4 2 2 0.5acidophilus 0681 Lactobacillus casei 1722  393 2 2 2 0.5 Lactobacillusplantarum 39268 2 2 2 0.5 2791 Peptostreptococcus — 2 8 2 1 anaerobius3526 Peptostreptococcus — 4 16 4 2 anaerobius 3531 Peptostreptococcus —0.25 0.25 0.12 0.06 micros 3432 Peptostreptococcus — 1 1 0.5 0.25 micros3545 Propionibacterium acnes — 0.25 0.25 0.12 0.06 1713Propionibacterium acnes 11829 1 1 0.5 0.5 1286 Streptococcus 2782332 >16 16 16 constellatus 1202 Streptococcus 27335 1 2 0.5 0.25intermedius 1203 Bacteroides fragilis 3374 — 0.12 0.5 0.12 0.03Bacteroides fragilis 3479 — 16 >16 16 8 Bacteroides ovatus 3503 — 8 >168 4 Bacteroides ovatus 3508 — 0.25 0.5 0.12 0.03 Bacteroides — 0.25 10.25 0.03 thetaiotaomicron 3399 Bacteroides — 16 >16 16 8thetaiotaomicron 3496 Bacteroides vulgatus — 16 >16 8 8 3389 Bacteroidesvulgatus — 16 >16 8 8 3494 Eikenella corrodens 43278 1 0.5 0.12 0.031206 Fusobacterium 25286 0.25 0.5 0.5 0.06 necrophorum 3963Fusobacterium 25586 0.25 0.5 0.5 0.06 nucleatum 3962 Porphyromonas —16 >16 4 8 asaccharolytica 3552 Porphyromonas — 8 16 2 4 asaccharolytica3557 Prevotella — 32 >16 16 16 melaninogenica 3437 Prevotella — 4 8 1 1melaninogenica 3443 Prevotella spp. 3564 — 4 1 1 0.25 Prevotella spp.3568 — 2 4 1 0.25 Veillonella parvula 1272 17745 4 1 1 0.5 “TET” istetracycline; “DOX” is doxycycline; “MIN” is minocycline; “ATCC” isAmerican Type Culture Collection.

Antibacterial Spectrum of Activity

An assessment of the antibacterial spectrum of activity of the activewas determined in several studies by in vitro MIC determination for avariety of Gram-positive and Gram-negative aerobic and anaerobicorganisms. The results of these assays (summarized in Table 7) indicatethat the active demonstrates activity against propionibacteria and otherGram-positive organisms with a narrower spectrum of activity thanclinically-used tetracyclines. Strains resistant to tetracycline arecross-resistant to the active. The activity for each organism group isdiscussed in the text that follows the table.

TABLE 7 Summary of In Vitro MIC testing against propionibacteria andaerobic Gram-positive and Gram-negative organisms MIC Organism [Type](No. Range MIC₅₀ MIC₉₀ isolates) Compound (μg/mL) (μg/mL) (μg/mL)Propionibacteria P. acnes The Active 0.25-4   0.5 2 [tetS] Tetracycline0.5-4   0.5 4 (13) Doxycycline 0.25-1   0.25 1 Minocycline ≤0.06->8  0.125 1 P. acnes The Active >8->8 [tetR] Tetracycline >8->8 (2)Doxycycline 8-8 Minocycline 2-2 P. acnes The Active 0.5-16  0.5 4[clinical isolates] Tetracycline 0.5-32  1 2 (55) Doxycycline 0.25-16  0.5 2 Minocycline 0.125-8    0.25 1 Clindamycin ≤0.06-64   ≤0.06 4Erythromycin ≤0.06->128  ≤0.06 >128 P. acnes The Active 0.25-1   [tetS]Tetracycline 0.25-1   (2) Doxycycline 0.12-0.5  Minocycline 0.06-0.5 Clindamycin 0.06-0.25 Metronidazole >32->32 Penicillin 0.03-0.5 Vancomycin 0.5-0.5 P. granulosum The Active 1-1 [clinical isolates]Tetracycline 1-2 (3) Doxycycline 0.5-1   Minocycline 0.25-0.5 Clindamycin ≤0.06-≤0.06 Erythromycin ≤0.06-≤0.06 P. avidum The Active1-4 [clinical isolates] Tetracycline 1-8 (4) Doxycycline 0.5-4  Minocycline 0.25-2   Clindamycin ≤0.06-0.5   Erythromycin 0.125-0.125Gram-positive aerobic bacteria S. aureus The Active ≤0.06-0.25   0.1250.25 [tetS] Tetracycline ≤0.06-0.25   0.25 0.25 (20) Doxycycline≤0.06-0.25   ≤0.06 0.25 Minocycline 0.125-0.5  0.25 0.5 S. aureus TheActive 0.125-32   4 16 [tetR] Tetracycline  2-64 64 64 (10) Doxycycline 1-16 4 16 Minocycline 0.25-16   0.5 8 S. epidermidis The Active0.12-2   0.25 2 [MSSE] Tetracycline 0.12-2   0.25 2 (31) Doxycycline0.06-1   0.12 1 Minocycline 0.06-0.25 0.06 0.25 Erythromycin 0.12->32 0.25 >32 Clindamycin ≤0.03->32  0.12 >32 Oxacillin 0.06-0.25 0.12 0.25Vancomycin 1-2 2 2 S. epidermidis The Active 0.25-2   0.5 2 [MRSE]Tetracycline 0.25->32  1 2 (32) Doxycycline 0.12-8   0.5 1 Minocycline0.06-0.5  0.12 0.25 Erythromycin 0.12->32  >32 >32 Clindamycin0.06->32  >32 >32 Oxacillin  0.5->32 32 >32 Vancomycin 1-2 2 2 S.pneumoniae The Active ≤0.06-0.125  [tetS] Tetracycline ≤0.06-0.25   (5)Doxycycline ≤0.06-0.125  Minocycline 0.25-0.25 S. pneumoniae The Active 4-32 [tetR] Tetracycline 32-64 (5) Doxycycline 4-4 Minocycline  8-16 S.pneumoniae The Active ≤0.03-32   0.12 0.25 [PSSP] Tetracycline 0.06->32 0.12 0.25 (32) Doxycycline 0.03->16  0.06 0.12 Minocycline ≤0.015->16  0.06 0.12 Erythromycin ≤0.015->16   0.03 2 Clindamycin ≤0.015->16   0.030.06 Penicillin ≤0.015-0.12   ≤0.015 0.06 Vancomycin 0.06-0.25 0.25 0.25S. pyogenes The Active 0.12-16   0.12 8 (32) Tetracycline 0.12-32   0.1232 Doxycycline 0.06-8   0.12 4 Minocycline 0.03-8   0.06 8 Erythromycin0.03->16  0.06 0.06 Clindamycin 0.03->16  0.03 0.06 Penicillin≤0.015-0.25   ≤0.015 ≤0.015 Vancomycin 0.25-0.5  0.25 0.25 S. pyogenesThe Active ≤0.06-0.25   [tetS] Tetracycline ≤0.06-0.125  (5) Doxycycline≤0.06-0.125  Minocycline 0.25-0.5  S. pyogenes The Active  4-16 [tetR]Tetracycline 32-64 (5) Doxycycline 4-8 Minocycline 4-8 S. agalactiae TheActive 0.12-32   16 16 (31) Tetracycline 0.12->32  32 >32 Doxycycline0.06-16   8 16 Minocycline 0.03-16   16 16 Erythromycin 0.03->16 0.06 >16 Clindamycin 0.03->16  0.06 >16 Penicillin ≤0.015-2    0.03 1Vancomycin 0.25-2   0.5 0.5 S. agalactiae The Active 0.125-0.25  [tetS]Tetracycline 0.25-0.25 (3) Doxycycline 0.25-0.25 Minocycline 0.5-0.5 S.agalactiae The Active 16-32 [tetR] Tetracycline 16-64 (7) Doxycycline 8-16 Minocycline  8-16 S. haemolyticus The Active 0.12-2   0.12 2 (33)Tetracycline 0.12->32  1 >32 Doxycycline 0.06-16   0.5 16 Minocycline≤0.03-0.5   0.06 0.5 Erythromycin 0.12->32  >32 >32 Clindamycin0.06->32  0.12 1 Oxacillin 0.06->32  0.25 >32 Vancomycin 0.5-2   1 1Streptococcus The Active 0.12-16   0.25 16 spp. Tetracycline 0.12->32 0.25 32 [Group C] Doxycycline 0.06-16   0.12 8 (30) Minocycline 0.03-8  0.06 8 Erythromycin ≤0.015->16   0.06 4 Clindamycin ≤0.015->16   0.060.12 Penicillin ≤0.015-0.03   ≤0.015 ≤0.015 Vancomycin 0.25-1   0.25 0.5E. faecalis The Active ≤0.06-≤0.06 [tetS] Tetracycline 0.25-0.5  (4)Doxycycline <0.06-0.125 Minocycline 0.25-0.5  E. faecalis The Active 8-32 [tetR] Tetracycline 32-64 (6) Doxycycline  2-16 Minocycline  4-16E. faecalis The Active 0.25-32   32 32 [VSE] Tetracycline 0.25->64  3264 (31) Doxycycline 0.12-16   8 8 Minocycline 0.06-16   8 16Erythromycin 0.25->32  >32 >32 Clindamycin  4->32 >32 >32 Ampicillin0.5-8   1 1 Vancomycin 0.5-4   1 2 E. faecium The Active ≤0.06-≤0.06[tetS] Tetracycline 0.125-0.25  (4) Doxycycline ≤0.06-≤0.06 Minocycline0.25-0.5  E. faecium The Active  8-32 [tetR] Tetracycline 32-64 (6)Doxycycline  4-16 Minocycline  2-32 E. faecium The Active 0.12-32   0.532 [VSE] Tetracycline 0.12->64  1 >64 (32) Doxycycline 0.06-32   0.5 16Minocycline ≤0.03-16   0.12 16 Erythromycin 0.06->32  >32 >32Clindamycin 0.12->32  >32 >32 Ampicillin 0.12->64  64 >64 Vancomycin0.25-2   1 1 E. faecium The Active 0.12-32   2 32 [VRE] Tetracycline0.12->64  2 >64 (30) Doxycycline 0.06-16   1 8 Minocycline ≤0.03-16  0.25 16 Erythromycin 0.12->32  >32 >32 Clindamycin 0.06->32  >32 >32Ampicillin  8->64 >64 >64 Vancomycin >64 >64 >64 Gram-negative aerobicbacteria E. coli The Active  4-32 [tetS] Tetracycline 1-4 (7)Doxycycline 0.5-4   Minocycline 0.5-4   E. coli The Active >64->64[tetR] Tetracycline >64->64 (3) Doxycycline 64-64 Minocycline  8-16 E.coli The Active  2->64 16 >64 (33) Tetracycline  1->64 2 >64 Doxycycline 0.5->32 2 32 Minocycline 0.25->32  1 8 Ampicillin  1->64 >64 >64Ciprofloxacin 0.008->2   0.015 >2 Cephalothin  2->64 32 >64 Tmp/Sxt≤0.06/1.19->64/1216   0.25/4.75  >64/1216 K. pneumoniae The Active 16-64[tetS] Tetracycline 0.5-4   (7) Doxycycline 0.5-8   Minocycline  1-16 K.pneumoniae The Active >64->64 [tetR] Tetracycline  8->64 (5) Doxycycline16-64 Minocycline  16->64 K. pneumoniae The Active  16->64 >64 >64 (31)Tetracycline  1->64 8 >64 Doxycycline  1->32 8 >32 Minocycline  1->324 >32 Ampicillin >64 >64 >64 Ciprofloxacin 0.03->2   >2 >2Cephalothin >64 >64 >64 Tmp/Sxt 0.12/2.38->64/1216 >64/1216 >64/1216 E.cloacae The Active 0.25->64  32 >64 (30) Tetracycline  0.5->64 2 >64Doxycycline 0.06->32  2 32 Minocycline ≤0.03->32  1 16 Ampicillin  4->6464 >64 Ciprofloxacin 0.008->2   0.25 >2 Cephalothin  2->64 >64 >64Tmp/Sxt ≤0.06/1.19->64/1216   0.25/4.75  >64/1216 P. mirabilis TheActive >64 >64 >64 (30) Tetracycline  16->64 32 64 Doxycycline 32->32 >32 >32 Minocycline  8->32 16 >32 Ampicillin  0.5->64 4 >64Ciprofloxacin 0.015->2   >2 >2 Cephalothin  2->64 8 >64 Tmp/Sxt≤0.06/1.19->64/1216   2/38 >64/1216 P. aeruginosa The Active 32->64 >64 >64 (11) Tetracycline  4->64 64 64 Doxycycline 4->32 >32 >32 Minocycline  8->32 >32 >32 Ampicillin >64 >64 >64Ciprofloxacin 0.12->2   >2 >2 Cephalothin >64 >64 >64 Tmp/Sxt  2/38->64/1216 16/304 >64/1216 Salmonella The Active  8->64 16 >64 spp.Tetracycline  1->64 2 >64 (35) Doxycycline  2->32 2 32 Minocycline 1->32 2 8 Ampicillin  0.5->64 1 >64 Ciprofloxacin 0.015-0.25  0.0150.03 Cephalothin  1->64 2 16 Tmp/Sxt ≤0.06/1.19->64/1216  ≤0.06/1.19->64/ 0.12/2.38  1216 Abbreviations used in Table 7: tetS,tetracycline sensitive; tetR, tetracycline resistant; VSE,vancomycin-susceptible Enterococcus; VRE, vancomycin-resistantEnterococcus; MSSE, methicillin-susceptible Staphylococcus epidermidis;MRSE, methicillin-resistant Staphylococcus epidermidis; PSSP,penicillin-susceptible Streptococcus pneumoniae; MIC, minimum inhibitoryconcentration; MIC₅₀, MIC at which 50% of isolates are inhibited; MIC₉₀,MIC at which 90% of the isolates are inhibited.

In Vitro Antibacterial Activity of the Active Against Propionibacteria

The in vitro antimicrobial activity of the active against the acnevulgaris pathogen P. acnes was assessed using the Clinical andLaboratory Standards Institute (CLSI)-approved agar dilution method foranaerobes. Susceptibility testing was performed by measuring the MICagainst a screening panel of P. acnes including severalmacrolide-resistant strains. The values determined for the active werecompared with similar members of the tetracycline class of antibiotics,including the clinically-used acne therapeutics, doxycycline andminocycline (Table 7). Against a panel of 13 tetracycline-sensitive P.acnes, the active demonstrated MICs comparable to doxycycline andminocycline.

In an expanded study, the active was tested against 62 recent clinicalisolates of propionibacteria along with several tetracycline comparators(Table 7). Against 55 strains of P. acnes isolated within the past 7years, including 26 isolates obtained within the past 3 years, theactive demonstrated activity similar to that of doxycycline.

In Vitro Antimicrobial Activity of the Active Against Staphylococci

Thirty strains of S. aureus were tested against the active (Table 7) andconventional tetracyclines (tetracycline, doxycycline, and minocycline).Typed strains with known resistance mechanisms, ribosomal protection andactive efflux, were included. Activity was variable and compared todoxycycline and minocycline.

In Vitro Antimicrobial Activity of the Active Against Streptococci

Thirty strains of streptococci (10 each S. pyogenes, S. agalactiae, andS. pneumoniae) were tested against the active and conventionaltetracyclines (tetracycline, doxycycline, and minocycline). Typedstrains with known resistance to tetracycline and minocycline,characteristic of ribosomal protection mediated by tetM or tetO, wereincluded. Similar to staphylococci, MIC ranges and MIC₉₀ values (Table7) were closest to minocycline and doxycycline. The active showed goodactivity against susceptible strains of S. pyogenes, with MICscomparable to doxycycline and minocycline.

The active demonstrated a bimodal distribution of MIC values for S.agalactiae with 7 strains inhibited at 0.25 μg/mL or less, and theremainder of the isolates requiring 16-32 μg/mL for inhibition. Theelevated MIC values tracked with resistance to tetracycline. The MIC₅₀and MIC₉₀ values for the active were similar to those of doxycycline andminocycline but less than that of tetracycline.

The active demonstrated potent activity against PSSP with all but 3strains inhibited at 0.25 μg/mL or less. The elevated MIC values trackedwith resistance to tetracycline. The activity was similar to that oftetracycline, doxycycline and minocycline.

The active demonstrated potent activity against S. pyogenes with all but6 strains inhibited at 0.25 μg/mL or less. Elevated MICs (≥4 μg/mL) wereobserved for 5 strains which tracked with resistance to tetracycline.The MIC₉₀ value for the active was similar to those of doxycycline andminocycline but less than that of tetracycline.

The active demonstrated a bimodal distribution of MIC values for Group Cstreptococci with 19 strains inhibited at 0.25 μg/mL or less, and theremainder of the isolates requiring 4-16 μg/mL for inhibition. Theelevated MIC values tracked with resistance to tetracycline. The MIC₅₀and MIC₉₀ values for the active were similar to those of tetracyclinebut higher than those of doxycycline and minocycline.

In Vitro Antimicrobial Activity of the Active Against Enterococci

Twenty enterococcal strains (10 each E. faecalis and E. faecium) weretested against the active and conventional tetracyclines (tetracycline,doxycycline, and minocycline), including 11 well characterizedtetracycline-resistant strains (Table 7). Unlike the staphylococci, theactive was not active against any tetracycline-resistant strains byefflux (mediated by tetK or tetL) or by ribosomal protection (tetM ortetO). The active did show activity against tetracycline-susceptiblestrains of E. faecalis and E. faecium which was comparable to that ofdoxycycline.

The active demonstrated a bimodal distribution of MIC values forvancomycin-susceptible E. faecalis with 8 strains inhibited at 0.5 μg/mLor less, and the remainder of the isolates requiring 16-32 μg/mL forinhibition. The elevated MIC values tracked with resistance totetracycline. The MIC₅₀ and MIC₉₀ values for the active were similar tothose of tetracycline and greater than those of doxycycline andminocycline.

The active also demonstrated bimodal distribution of MIC values forvancomycin-susceptible E. faecium with 17 strains inhibited at 0.5 μg/mLor less, and the remainder of the isolates requiring 16-32 μg/mL forinhibition. The elevated MIC values tracked with resistance totetracycline. The MIC₅₀ and MIC₉₀ values for the active were similar tothose of doxycycline and minocycline with an MIC₉₀ value lower than thatof tetracycline.

The active demonstrated a broad range of MIC values forvancomycin-resistant E. faecium. The elevated MIC values tracked withresistance to tetracycline. The MIC₅₀ value for the active was similarto that of tetracycline and doxycycline and 8-fold higher than that ofminocycline. The MIC₉₀ value for the active was lower than that oftetracycline and greater than that of doxycycline and minocycline. Ahigh percentage of strains were resistant to the rest of the testagents.

In Vitro Antimicrobial Activity Against Gram-Negative Bacteria

Against 7 tetracycline-sensitive strains of E. coli, the active was lessactive in vitro than doxycycline and minocycline (Table 7). Even lessactivity for the active was observed against tetracycline-sensitivestrains of K. pneumoniae. In contrast, doxycycline and minocyclinedemonstrated greater activity against these organisms than the active.As expected, no activity for the active was observed against 6tetracycline resistant strains demonstrating active efflux mediated bytetB or tetD.

The active had generally poor activity against E. cloacae though a smallnumber of strains (7 of 30) were inhibited at 1 μg/mL or less. The MIC₅₀value was 16- to 32-fold higher than those of the other tetracyclines.The MIC₉₀ value for the active was the same as tetracycline and higherthan that of doxycycline or minocycline.

The active was the least active of the tetracyclines against E. coliwith MIC₅₀ and MIC₉₀ values of 16 and >64 μg/mL, respectively. Theactive was the least active of the tetracyclines against K. pneumoniae.The active had generally poor activity against Salmonella spp. and wasthe least active of the tetracyclines.

Mechanism of Action

For the studies below, samples were prepared with bis hydrochloridesalt, and the data is expressed based on the free base (“the active”).The mechanism of action of the Crystalline Mono Hydrochloride Salt wasdetermined by two different approaches via study of the active, asdescribed below.

In the first approach, Antibacterial Mechanism of Action: In VitroInhibition of Bacterial Transcription and Translation, the ability ofthe active to inhibit bacterial protein synthesis was assessed using anin vitro cell-free bacterial transcription and translation assay(commercially-available from Promega Corporation, Madison, Wis.)(Beckler, G., Promega Notes 31 (1991) pp. 3-6). The active inhibited thesynthesis of reporter protein with an IC₅₀ of 8.3±0.18 μM. This valuewas comparable to the IC₅₀ values determined for the comparatortetracyclines, doxycycline and minocycline (IC₅₀ values of 4.7±0.48 and2.4 f 0.22 μM, respectively). These results provide evidence that theactive functions as a classical tetracycline by inhibiting bacterialprotein synthesis.

In the second approach, Antibacterial Mechanism of Action: Inhibition ofMacromolecular Synthesis in Staphylococcus aureus, the ability of theactive to target bacterial protein synthesis was further confirmed in awhole-cell assay of macromolecular synthesis in the Gram-positiveorganism, S. aureus. The active inhibited, in a dose-dependent manner,the incorporation of [³H]-leucine into proteins of the growing organismwithin the concentration range of 0.25-8 fold the MIC (0.063-2 μg/mL). Amaximum inhibition of 80% was observed at 8-fold the MIC which wascomparable to the values obtained for the tetracycline comparatorsdoxycycline and minocycline. In contrast, the active at 8-fold the MICdemonstrated less than 20% inhibition for the synthesis of cell wall,DNA, RNA and lipid components of the test bacteria. The results of thisstudy indicate that the active acts as a selective inhibitor ofbacterial protein synthesis at concentrations comparable to knowntetracyclines.

The in vitro susceptibility studies described above includedtetracycline-resistant strains with characterized tetracyclineresistance genes. Strains were selected that harbored the most commontetracycline resistance genes: efflux (tetK, tet38, tetL, tetS, tetB,and tetD), ribosomal protection (tetM and tetO), as well as P. acnesresistant by rRNA point mutation. The MIC values for these selectedstrains demonstrated a degree of cross-resistance between the active andother tetracyclines, as shown in Table 8. The presence of a tetracyclineresistance gene increased the MIC of the active relative to susceptiblestrains (with the exception of tetK in S. aureus), with MIC valuessimilar to those of doxycycline and/or minocycline, but generally lowerthan those of tetracycline.

TABLE 8 Activity of the Active Against Bacterial Strains withCharacterized Tetracycline Resistance Mechanisms. The Strain ActiveDoxycycline Minocycline PBS Mech./ MIC MIC MIC Organism # Genotype(μg/mL) (μg/mL) (μg/mL) P. acnes 1073 16S rRNA >8 8 2 point mutation S.aureus 1739 tet38 4 2 0.5 E. coli 669 tetB >64 64 16 K. pneumoniae 266tetD >64 64 64 S. aureus 1309 tetK 0.5 2 0.5 E. faecium 1323 tetK 8 4 2E. faecalis 274 tetL 32 16 16 S. aureus 1310 tetM 8 16 4 S. pyogenes 792tetM 4 4 4 S. agalactiae 897 tetM 16 8 16 S. pneumoniae 511 tetM 4 4 8E. faecalis 276 tetM 16 8 16 E. faecium 965 tetM 8 4 8 S. pyogenes 330tetO 16 8 8 S. agalactiae 316 tetO 32 8 16 E. faecium 1324 tetO 16 4 2E. faecalis 949 tetS 8 2 4

Animal Models of Infection

For the studies below, samples were prepared with bis hydrochloridesalt, and the data is expressed based on the free base (“the active”).In vivo efficacy studies were conducted with the active in threedistinct animal infection models and one inflammation model. Bypossessing comparable activity to the active, the studies show: 1)anti-infective efficacy of the Crystalline Mono Hydrochloride Saltcompared to other commercially available tetracycline acne medications(doxycycline and minocycline) against representative Gram-positivepathogens with similar in vitro susceptibility as P. acnes; and 2) theanti-inflammatory activity of Crystalline Mono Hydrochloride Salt.

The comparators were dosed at a concentration of 10 mg/mL in a vehicleof sterile water and adjusted for percent of their active moiety. Allstudies, except for the thigh wound and rat footpad edema studies, wereconducted in CD-1 male mice. The thigh wound model was conducted usingCD-1 female mice and the rat footpad edema studies were conducted withSprague-Dawley male rats.

Table 9 presents the data collected from the following three studies,which evaluated the efficacy of the active.

The first study evaluated the active in an S. aureus SystemicIntraperitoneal Challenge (IPC) Model. The objective was to assess thein vivo activity of the active against a Gram-positive organism in anacute infectious model compared to commercially-available tetracyclinetreatments for acne vulgaris. Bacterial cultures were prepared bygrowing tetracycline-sensitive S. aureus RN450-1 overnight, thendiluting with sterile phosphate-buffered saline (PBS). For eachexperiment, a total of 30 mice were infected by injection into theintraperitoneal cavity with 500 μl of 7.5×10⁷ CFU in 5% sterilebacteriological mucin. Four to five treatment groups of 5 mice each weretreated with a single injection of the active, doxycycline, and/orminocycline at doses ranging from 0.01-0.5 mg/kg in sterile water. Thedoses were administered subcutaneously (SC) at 1 hour post infection. Anadditional infected group of 5 mice was included as a negative control(untreated) group. For all studies but one, a positive control group(s)was included (e.g., doxycycline, minocycline, or ciprofloxacin). Micewere monitored for survival for up to 7 days. Efficacy was determined bycalculating the PD₅₀ at 48 hours post infection. PD₅₀ (protective dose,50%) is the dose required to achieve 50% survival. The PD₅₀ valuesreported are a mean of 2-3 independent experiments for each drug tested.

The active displayed potent activity against S. aureus RN450-1 resultingin a PD₅₀ of 0.25 mg/kg and demonstrated activity similar to doxycyclineagainst a representative Gram-positive pathogen.

The second study, Efficacy of the Active in a S. aureus Thigh WoundInfection Model, looked at the activity of the active against arepresentative Gram-positive infection in a tissue-based infectionmodel. The efficacy of the active was studied against S. aureus RN450-1in a thigh wound model of immunocompromised mice. A total of 40 mice(n=4-8 mice per group) were rendered neutropenic by cyclophosphamidetreatment four days before (150 mg/kg) and one day before (100 mg/kg)infection. Bacterial cultures were prepared by growing S. aureus RN450-1overnight and diluting with sterile PBS. An injection of 100 μL of˜1×10⁶ CFU/mL in sterile PBS was injected into the thigh. Four groups ofmice for each drug received doses of 0.33, 1, 3, or 9 mg/kgintravenously at 2 and 6 hours post infection. An additional group ofuntreated mice served as a negative control group. At 24 hours postinfection, the thighs were collected aseptically, homogenized, andplated to enumerate bacterial load per thigh. Data are reported as ED₅₀values. ED₅₀ (effective dose, 50%) is the dose required to achieve a 2log₁₀ reduction in bacterial burden (colony forming units [CFU]/wholeorgan) in the target organ compared to untreated controls.

As shown in Table 9, the active demonstrated efficacy equivalent todoxycycline, a commonly used treatments for acne vulgaris, in the S.aureus thigh wound model.

The third study, Efficacy of the Active in an Acute S. pneumoniaeRespiratory Tract Infection (RTI) Model, demonstrated activity of theactive in an additional tissue-based infection model compared todoxycycline. The active and doxycycline were tested independentlyagainst an S. pneumoniae-induced acute pneumonia in immunocompromisedmice. In each experiment, 35 mice were rendered neutropenic by anintraperitoneal (IP) injection of cyclophosphamide four days before (150mg/kg) and one day before (100 mg/kg) infection. Tetracycline-sensitiveS. pneumoniae PBS 1339 was grown on plates overnight, coloniescollected, and resuspended in sterile PBS. Mice were infectedintra-nasally with 50 μL of this bacterial suspension containing ˜6×10⁶CFU/mouse. At 2 hours post infection, 4 to 5 groups of 5 mice each weretreated with a single intravenous (IV) dose of the active or doxycyclineat 5 (the active only), 10, 20, 40, or 80 mg/kg dissolved in sterilewater. Each study also had an untreated control group and a group thatreceived a positive control compound (e.g., vancomycin at 20 mg/kg IV).Efficacy was determined by calculating the PD₅₀ at 72 hours postinfection.

A single IV dose of the active exhibited activity in a neutropenic,lethal S. pneumoniae RTI model at a PD₅₀ of 4.66 mg/kg (as shown inTable 9). This dose was slightly lower than the 7.18 mg/kg dose requiredto achieve the PD₅₀ for doxycycline. The active demonstrated activity inthis additional tissue-based infection model that was comparable to, orslightly better than, that of doxycycline.

TABLE 9 Efficacy Summary of the Active and Comparators in MurineInfection Models. S. aureus S. aureus S. pneumoniae RN450-1 IPC RN450-1Thigh PBS1339 RTI Compound PD₅₀ (mg/kg) ED₅₀ (mg/kg) PD₅₀ (mg/kg) TheActive 0.25 8.23 4.66 Doxycycline 0.30 8.31 7.18 Minocycline 0.03 — —

The study, In Vivo Efficacy of the Active in an Inflammation Model ofRat Carrageenan-Induced Footpad Edema, was conducted to evaluate theanti-inflammatory properties of the active compared to minocycline anddoxycycline. Groups of 3-8 rats were injected IP with the active,doxycycline, minocycline, and/or saline control at 5 minutes precedingan injection of the inflammatory carrageenan solution (1 mg/100 μL) inthe hind paw. Each study also had a saline treated control group (3-8rats/group). The active was tested at 5, 10, 25, 50, 75, 100, or 150mg/kg. Minocycline was tested at 25, 50, 75, or 100 mg/kg anddoxycycline was tested at 75 and 100 mg/kg. Immediately following thecarrageenan injection and 3 hours post-injection, the hind paw volumewas measured with a digital water plethysmometer. Results werecalculated as a percent change in paw volume over the 3 hours, dividedby the baseline paw volume, and then adjusted for the mean percentinflammation in the untreated controls, and are presented in Table 10.The active, doxycycline, and minocycline all demonstratedanti-inflammatory activity at all doses tested. The active exhibitedanti-inflammatory activity in a standard animal model of inflammationcomparable to other commercially-available tetracyclines commonly usedfor the treatment of acne vulgaris.

TABLE 10 Mean inhibition of Inflammation by the Active in a Carrageenan-Induced Rat Footpad Edema Model. Mean Percent Inflammation Compared toUntreated Controls Compound 150 mg/kg 100 mg/kg 75 mg/kg 50 mg/kg 25mg/kg 10 mg/kg 5 mg/kg 1 mg/kg The Active 26 53 56 52 59 65 78 103Doxycycline — 36 68 — — — — — Minocycline — 21 54 33 47 — — —

Numerous alterations, modifications, and variations of the preferredembodiments disclosed herein will be apparent to those skilled in theart, and they are all anticipated and contemplated to be within thespirit and scope of the claimed invention. For example, althoughspecific embodiments have been described in detail, those with skill inthe art will understand that the preceding embodiments and variationscan be modified to incorporate various types of substitute, additionalor alternative materials. Accordingly, even though only few variationsof the present invention are described herein, it is to be understoodthat the practice of such additional modifications and variations andthe equivalents thereof, are within the spirit and scope of theinvention as defined in the following claims. All patent applications,patents, and other publications cited herein are incorporated byreference in their entirety.

What is claimed is:
 1. A crystalline salt of(4S,4aS,5aR,12aS)-4-dimethylamino-3,10,12,12a-tetrahydroxy-7-[(methoxy(methyl)amino)-methyl]-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydro-naphthacene-2-carboxylicacid amide, wherein the salt is selected from a group consisting of monohydrochloride, mono mesylate and mono sulfate.
 2. The crystalline saltof claim 1, wherein the salt is substantially pure.
 3. The crystallinesalt of claim 1, wherein the salt is mono hydrochloride.
 4. Thecrystalline salt of claim 3, having an XRPD pattern substantially asillustrated in FIG. 1 after synthesis of the crystalline salt.
 5. Thecrystalline salt of claim 3, having characteristic peaks at diffractionangle 2-theta degrees appearing at least at about 13.4, about 20.5 andabout 23.3, as measured by XRPD.
 6. The crystalline salt of claim 3,having a DSC curve substantially as illustrated in FIG. 2 aftersynthesis of the crystalline salt.
 7. The crystalline salt of claim 3,having a TGA curve substantially as illustrated in FIG. 3 aftersynthesis of the crystalline salt.
 8. The crystalline salt of claim 3,wherein the salt has a J-isomer content at 0 days of about 0.1% peakarea to about 7.0% peak area, as measured by HPLC.
 9. The crystallinesalt of claim 1, wherein the salt is mono mesylate.
 10. The crystallinesalt of claim 9, having an XRPD pattern substantially as illustrated inFIG. 4 after synthesis of the crystalline salt.
 11. The crystalline saltof claim 9, having characteristic peaks at diffraction angle 2-thetadegrees appearing at least at about 9, about 15 and about 23.8, asmeasured by XRPD.
 12. The crystalline salt of claim 9, having a DSCcurve substantially as illustrated in FIG. 5 after synthesis of thecrystalline salt.
 13. The crystalline salt of claim 9, having a TGAcurve substantially as illustrated in FIG. 6 after synthesis of thecrystalline salt.
 14. The crystalline salt of claim 9, wherein the salthas a β-isomer content at 0 days of about 2.0% peak area to about 10.0%peak area, as measured by HPLC.
 15. The crystalline salt of claim 1,wherein the salt is mono sulfate.
 16. The crystalline salt of claim 15,having an XRPD pattern substantially as illustrated in FIG. 7 aftersynthesis of the crystalline salt.
 17. The crystalline salt of claim 15,having characteristic peaks at diffraction angle 2-theta degreesappearing at least at about 15, about 17.8 and about 23.5, as measuredby XRPD.
 18. The crystalline salt of claim 15, having a DSC curvesubstantially as illustrated in FIG. 8 after synthesis of thecrystalline salt.
 19. The crystalline salt of claim 15, having a TGAcurve substantially as illustrated in FIG. 9 after synthesis of thecrystalline salt.
 20. The crystalline salt of claim 15, wherein the salthas a β-isomer content at 0 days of about 3.0% peak area to about 26.0%peak area, as measured by HPLC.